Plasmon absorption in metal nanoparticles is highly dependent on nanoparticle shape, size, and dielectric constant of the surrounding medium. Strong plasmon absorption and sensitivity to local environment have made metal nanoparticles attractive candidates as sensors for a range of analytes including DNA, metal ions, and antibodies.
Much of the work in this area has focussed on the use of suspensions of metals, especially “noble” metals such as gold or silver, gold being particularly suitable in view of its biocompatibility.
For example, in WO 2006/137851 (Virginia Tech Intellectual Properties Inc.) the preparation of a metallic suspension by the direct coating of silica nanoparticles by gold or silver is reported. A method is described of making a metallic suspension, involving the activation of silica particles in a silica colloidal suspension; the nucleation of noble metal atoms to the silica particles in the suspension and the subsequent growth of noble metal particles on the silica particles at the nucleation sites.
WO 2006/099312 (North-Western University) relates to a Method of Producing Gold Nanoprisms and is also concerned with metallic suspensions.
WO 2006/065762 (University of South Carolina) and WO 2006/066180 (Intel Corporation) relate to Surface Enhanced Raman Spectroscopy (SERS) facilitated by the use of gold nanoparticles, though the materials provided employ continuous metallic surfaces.
A more powerful method of utilising the interesting photonic properties of nanoparticles would be to provide the nanoparticles in the form of a 2D photonic array.
In general a photonic biosensor array, sometimes called a microarray or biochip, comprises a collection of probe spots to which different targets may attach. For example in the case of a DNA microarray the probes are oligonucleotides, cDNA or similar which are hybridised with fluorescence labelled samples, typically of two colours, one for the patient the other for the control. Typically, fluorescence from the hybridised array is then viewed to determine to which spots binding has occurred. There are other types of array such as protein arrays (including antibody arrays) where spots of protein molecules (or antibodies) are used to identify the complementary entity (antibodies or proteins). Thus chemical compound arrays may be employed to search for proteins and other biologically active molecules again by employing functionalising molecules or entities in an array of spots which bind with specific biological targets. In general, however, all these techniques employ fluorescence labelling of the targets to detect binding events on the array.
The arrays provided by the methods of the present invention do not employ fluorescence but instead rely upon plasmon resonance-based sensing. Broadly speaking in this technique total internal reflection of light is used to generate an evanescent wave which excites plasmons (a collective electronic excitation) in a metallic conductor, which are modified by the presence of a target molecule on the surface of the conductor. The modification results in a shift, generally in both wavelength and amplitude, of the plasmon resonance peak detectable in the totally internally reflected light. Plasmon resonance-based sensing has the ability to detect very small changes in the effective refractive index in a medium adjacent the surface of the metallic conductor, for example down to Δn of the order of 10−4 refractive index units (RIU).
It is known to employ label-free surface plasmon resonance (SPR) based technology for studying biomolecular interaction in real time and, in particular, technology for this is available from the Swedish company BIAcore AB; for background technical information see published BIAcore patent applications such as WO 2006/135309, WO 94/00751, U.S. Pat. No. 4,997,278, and WO 97/19375. However BIAcore provide materials employing a continuous metal surface. Some further technical background information relating to plasmon resonance-based sensing in a different context (evanescent wave cavity ringdown spectroscopy) can be found in EvanesCo patent application WO 2005/088277.
There is, however, a need to provide an improved method of preparation of photonic arrays suitable for use in a wide variety of assay techniques, and in particular leading to assay techniques with increased sensitivity.
Embodiments of the techniques we describe provide a step towards solutions of these problems.